The long-term goal of this proposal is to understand the molecular, cellular and physiological mechanisms by which protein scaffolds coordinate signal propagation and signal termination in insulin sianalinq. Specifically, we focus on how protein scaffolds allow the activity of S6 kinase (S6K), a key metabolic transducer, to be enhanced by atvpical protein kinase C (aPKC) isozymes, promoting insulin sensitivity, and suppressed by the novel phosphatase PH domain Leucine-rich repeat Protein Phosphatase (PHLPP; pronounced 'flip'), promoting insulin resistance. The canonical pathway for transduction of the insulin signal is through the kinase Akt. This results in activation of S6K which, in liver, is associated with liponeogenesis. However, under conditions of high fat diet/obesity, insulin resistance occurs because a feedback loop from activated S6K dampens Akt signaling. We will test the hypothesis that aPKC activity dominates under high fat diet, promoting the phosphorylation of S6K at a key regulatory site, the hydrophobic motif, thus creating a bypass pathway to allow liponeogenesis when Akt activity is feedback- inhibited. We will also test the hypothesis that PHLPP, which dephosphorylates the hydrophobic motif of two other related kinases, directly dephosphorylates the hydrophobic motif of S6K. Furthermore, a central hypothesis driving this proposal is that coordination of atvpical PKC and PHLPP on protein scaffolds mediates their control of S6K activity. Three Aims are proposed: 1] to explore the mechanism by which the hydrophobic motif on S6K is under opposing regulation by the kinase aPKC and phosphatase PHLPP, 2] to test the hypothesis that aPKC is part of a bypass pathway that allows S6K to signal under conditions of high fat diet, and 3] to use mouse models to test the hypothesis that PHLPP is upregulated under high fat diet, suppressing S6K activity, and thus contributing to insulin resistant states. RELEVANCE: This project addresses the fundamental mechanisms in insulin resistance that accompanies diabetes and metabolic syndrome. With approximately one third of the US population considered obese, including an increasing proportion of children, these two diseases have reached epidemic proportions. Thus, understanding the molecule mechanisms of insulin resistance is essential for pharmacological intervention of the pathophysiologies associated with obesity.